Cucumber Hybrid Menfis

ABSTRACT

A hybrid cucumber designated Menfis is disclosed having resistance to powdery mildew ( Sphaerotheca fuliginea ), cucumber mosaic virus, and cucumber vein yellowing virus. The invention relates to the seeds of hybrid cucumber Menfis, to the plants of hybrid cucumber Menfis, and to methods for producing a cucumber plant, either inbred or hybrid, by crossing the hybrid Menfis with itself or another cucumber plant. The invention further relates to methods for producing a cucumber plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other cucumber lines, cultivars, or hybrids derived from the hybrid cucumber Menfis.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive cucumber hybriddesignated Menfis which is a parthenocarpic slicing cucumber that alsohas intermediate resistance to powdery mildew, cucumber mosaic virus,and cucumber vein yellowing virus. All publications cited in thisapplication are herein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The selected germplasm is crossed in order torecombine the desired traits and through selection varieties or parentlines are developed. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm. These important traits may include higher yield, fieldperformance, fruit and agronomic quality, such as fruit shape andlength, resistance to diseases and insects, and tolerance to drought andheat.

Practically speaking, all cultivated forms of cucumber belong to thehighly polymorphic species Cucumis sativus L. that is grown for itsedible fruit. As a crop, cucumbers are grown commercially whereverenvironmental conditions permit the production of an economically viableyield. They can be hand or mechanically harvested. Cucumbers that aregrown for fresh market, also called slicers, are generally handharvested. Those that are to be processed are called “picklers” and maybe hand or mechanically harvested. They are produced on trailing orclimbing vines. On healthy plants there is a canopy of large, regular,three-lobed leaves in an alternate arrangement. Pickling cucumbers grownin the United States have usually blunt and angular fruits. They arewhite-spined and most possess dark green or medium dark green exteriorcolor. Most slicers have slightly rounded ends and taper slightly fromthe stem to blossom end. However, cylindrical-shaped fruits with blockyor even rounded ends are also available.

Many changes that occurred with the domestication of the cucumber relateto fruit morphology, with a specialization in fruit shape and size.Slicing cucumbers are frequently sold in lengths from 15 cm to 25 cm anddiameter varies from 2.5 cm to nearly 7 cm. In the United States, theprincipal slicer cucumber growing regions are Georgia, Florida,Michigan, California, and North Carolina, with nearly 42,000 acres outof a U.S. total acreage of 57,500 acres. The main states that produceprocessing cucumbers are Michigan, North Carolina, and Texas. Freshcucumbers are available in the United States mainly from spring to fall.Cucumbers are consumed in many forms; generally processed for picklingtypes and as fresh market product for slicers. Although slicingcultivars may be processed, they generally are not acceptablesubstitutes for the pickling cucumbers.

Cucumis sativus is a member of the family Cucurbitaceae. TheCucurbitaceae is a family of about 90 genera and 700 to 760 species,mostly of the tropics. The family includes melons, pumpkins, squashes,gourds, watermelon, loofah, and many weeds. The genus Cucumis, to whichthe cucumber and several melons belong, includes about 70 species. Thecucumber is believed to be native to India or Southern Asia and has beencultivated there for about 3000 years.

Cucumber is distinct from other Cucumis species in that it has sevenpairs of chromosomes (2n=2x=14) whereas most others have twelve pairs ormultiples of twelve. Pollination techniques for controlled crosses incucumbers are easy to conduct. If bees and natural pollen vectors can beexcluded, the breeder need not be concerned about preventing selfing orother pollen contamination because of the diclinous nature of cucumbersand the stickiness or adherence of pollen to its source flower. There isno wind dissemination of pollen. Pistillate flowers are receptive in themorning or up to midday on the day they open. Cucumbers have a broadrange of floral morphologies, from staminate, pistillate tohermaphrodite flowers, yielding several types of sex expression.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three years. The best lines are candidatesfor new commercial cultivars. Those lines still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior because, for most traits, the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of cucumber plant breeding is to develop new, unique, andsuperior cucumber cultivars and hybrids. The breeder initially selectsand crosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing, and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same cucumber traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic, and soil conditions, and further selections arethen made during and at the end of the growing season. The cultivars orhybrids that are developed are unpredictable because the breeder'sselection occurs in unique environments with no control at the DNA level(using conventional breeding procedures), and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same cultivar twice by using the same originalparents and the same selection techniques. This unpredictability resultsin the expenditure of large amounts of research monies to developsuperior new cucumber cultivars and hybrids.

The development of commercial cucumber cultivars requires thedevelopment of cucumber parental lines, the crossing of these lines, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichlines are developed by selfing and selection of desired phenotypes. Thenew lines are crossed with other lines and the hybrids from thesecrosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population. Then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison, and characterization of plant genotype. Amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen (Molecular Linkage Map ofSoybean (Glycine max), pp. 6.131-6.138 in S. J. O'Brien (Ed.) GeneticMaps: Locus Maps of Complex Genomes, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1993)), developed a molecular geneticlinkage map that consisted of 25 linkage groups with about 365 RFLP, 11RAPD, three classical markers, and four isozyme loci. See also,Shoemaker, R. C., RFLP Map of Soybean, pp. 299-309, in Phillips, R. L.and Vasil, I. K. (Eds.), DNA-Based Markers in Plants, Kluwer AcademicPress, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. Diwan, N. and Cregan, P.B., Theor. Appl. Genet., 95:22-225 (1997). SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intocucumber varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid, or acridines. Once a desired trait isobserved through mutagenesis, the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company (1993).

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan, et al., Theor. Appl. Genet., 77:889-892 (1989).

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding, John Wiley and Son, pp.115-161 (1960); Allard (1960); Simmonds (1979); Sneep, et al. (1979);Fehr (1987); “Carrots and Related Vegetable Umbelliferae,” Rubatzky, V.E., et al. (1999)).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor, and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Cucumber is an important and valuable field crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding cucumberhybrids that are agronomically sound. To accomplish this goal, thecucumber breeder must select and develop cucumber plants that have thetraits that result in superior parental lines for producing hybrids.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a hybrid cucumber plantdesignated Menfis. This invention thus relates to the seeds of hybridcucumber Menfis, to the plants of cucumber Menfis, to methods forproducing a cucumber plant produced by crossing hybrid cucumber Menfiswith itself or another cucumber plant, and to methods for producing acucumber plant containing in its genetic material one or more transgenesand to the transgenic cucumber plants produced by that method. Thisinvention also relates to methods for producing other cucumber cultivarsor hybrids derived from hybrid cucumber Menfis and to the cucumbercultivars and hybrids derived by the use of those methods. Thisinvention further relates to cucumber seeds and plants produced bycrossing hybrid cucumber Menfis with another cucumber cultivar.

Parts of the cucumber plant of the present invention are also provided,for example, but not limited to, pollen, ovule, fruit, cells, embryos,meristems, cotyledons, leaves, anthers, roots, root tips, pistil,flower, seed, and stalks.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of hybrid cucumber plant Menfis. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoingcucumber plant, and of regenerating plants having substantially the samegenotype as the foregoing inbred cucumber plant. Preferably, theregenerable cells in such tissue cultures will be produced from embryo,protoplast, meristematic cell, callus, pollen, leaf, stem, petiole,root, root tip, fruit, seed, flower, anther, pistil, or the like. Stillfurther, the present invention provides cucumber plants regenerated fromtissue cultures of the invention.

Another objective of the invention is to provide methods for producingother cucumber plants derived from hybrid cucumber Menfis. Cucumbercultivars and hybrids derived by the use of those methods are also partof the invention.

The invention also relates to methods for producing a cucumber plantcontaining in its genetic material one or more transgenes and to thetransgenic cucumber plant produced by that method.

In another aspect, the present invention provides for single geneconverted plants of hybrid cucumber Menfis. The single transferred genemay preferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such trait as sex determination, herbicideresistance, insect resistance, resistance for bacterial, fungal, orviral disease, improved harvest characteristics, enhanced nutritionalquality, or improved agronomic quality. The single gene may be anaturally occurring cucumber gene or a transgene introduced throughgenetic engineering techniques.

The invention further provides methods for developing cucumber plants ina cucumber plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, and transformation. Marker loci, such asrestriction fragment polymorphisms or random amplified DNA, have beenpublished for many years and may be used for selection. See, Pierce, etal., HortScience, 25:605-615 (1990); Wehner, T., Cucurbit GeneticsCooperative Report, 20: 66-88 (1997); and Kennard, et al., TheoricalApplied Genetics, 89:217-224 (1994). Seeds, cucumber plants, and partsthereof produced by such breeding methods are also part of theinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Androecious plant. A plant having staminate flowers only.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Blossom end. The blossom end is the distal end of the fruit where theflower blossom is located (i.e., the “far” end as measured from the baseof the plant). The other end of a fruit is the stem end.

Blossom scar. The blossom scar is the small mark left on the distal endof the fruit after the flower falls off.

Blunt ends. Blunt ends are ends of the cucumber fruits that are nottapered or rounded.

Covered cultivation. Any type of cultivation where the plants are notexposed to direct sunlight. The covering includes, but is not limitedto, greenhouses, glasshouses, nethouses, plastic houses, and tunnels.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Gynoecious plant. A plant having pistillate flowers only.

Indeterminate vine or indeterminate growth. Refers to apical meristemproducing an unrestricted number of lateral organs; characteristic ofvegetative apical meristems. (Anatomy of Seed Plants, 2nd Edition, JohnWiley and Sons, p. 513 (1977). The main stem of the plant continues togrow as long as the plant stays healthy, as opposed to a determinateplant, which at some point in its life cycle will stop growing longer.

Monoecious plant. A plant having separate staminate and pistillateflowers on the same plant.

Parthenocarpic. “Parthenocarpic” refers to the ability of fruit todevelop without pollination or fertilization. The fruit are thereforeseedless.

Percent identity. Percent identity as used herein refers to thecomparison of the homozygous alleles of two cucumber lines, hybrids, orvarieties. Percent identity is determined by comparing a statisticallysignificant number of the homozygous alleles of two developed varieties,lines, or hybrids. For example, a percent identity of 90% betweencucumber plant 1 and cucumber plant 2 means that the two plants have thesame allele at 90% of their loci.

Percent similarity. Percent similarity as used herein refers to thecomparison of the homozygous alleles of a cucumber plant such as hybridcucumber Menfis with another plant, and if the homozygous allele ofhybrid cucumber Menfis matches at least one of the alleles from theother plant then they are scored as similar. Percent similarity isdetermined by comparing a statistically significant number of loci andrecording the number of loci with similar alleles as a percentage. Apercent similarity of 90% between hybrid cucumber Menfis and anotherplant means that hybrid cucumber Menfis matches at least one of thealleles of the other plant at 90% of the loci.

Propagate. To “propagate” a plant means to reproduce the plant by meansincluding, but not limited to, seeds, cuttings, divisions, tissueculture, embryo culture, or other in vitro method.

Quantitative trait loci (QTL). Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single gene transferred into the inbred via the backcrossingtechnique, genetic engineering, or mutation.

Transgene. A “transgene” is a gene taken or copied from one organism andinserted into another organism. A transgene may be a gene that isforeign to the receiving organism or it may be a modified version of anative, or endogenous, gene.

Vertical growing system. A “vertical growing system” means a plantgrowing technique in which plants are grown vertical to the ground withthe use of supporting material. The supporting material includes, but isnot limited to, wires or nets.

DETAILED DESCRIPTION OF THE INVENTION

Hybrid cucumber Menfis is a slicer cucumber with superiorcharacteristics. Hybrid cucumber Menfis is intended for coveredcultivation from spring to autumn. Hybrid cucumber Menfis is moderatelyresistant to powdery mildew (Podosphaera xanthii) and cucumber mosaicvirus (CMV), and is resistant to cucumber vein yellowing virus (CVYV).

Menfis is a cucumber hybrid with high yield potential and medium fruitwith dark-green skin. The hybrid has shown uniformity and stability forthe traits, within the limits of environmental influence for the traits.Hybrid cucumber Menfis has been increased with continued observation foruniformity. No variant traits have been observed or are expected inMenfis.

Hybrid cucumber Menfis has the following morphologic and othercharacteristics.

TABLE 1 VARIETY DESCRIPTION INFORMATION Type: Predominant usage: Freshmarket Predominant culture: Greenhouse Fruit type: American slicerPlant: Growing season: Winter Sex expression: Gynoecious Parthenocarpy:Present Ovary, color of vestiture: White Fruit (young): Type ofvestiture: Prickles only Fruit (at market maturity): Length: MediumPredominant shape of stem end: Acute Length of neck: Short Ground colorof skin: Green Intensity of ground color of skin: Dark Ribs: Absent orweak Cotyledon bitterness: Absent Disease Resistance: Cucumber scab(Cladosporium cucumerinum): Resistant Powdery mildew (Podosphaeraxanthii): Moderately resistant Corynespora blight and target spotSusceptible (Corynespora cassiicola): Cucumber mosaic virus (CMV):Moderately resistant Cucumber vein yellowing virus (CVYV): ResistantZucchini yellows mosaic virus (ZYMV): Susceptible Cucumber yellowstunting disorder virus: Intermediate resistant

Further Embodiments of the Invention

This invention also is directed to methods for producing a cucumberplant by crossing a first parent cucumber plant with a second parentcucumber plant wherein either the first or second parent cucumber plantis a hybrid cucumber plant of Menfis. Further, both first and secondparent cucumber plants can come from the hybrid cucumber Menfis. Allplants produced using hybrid cucumber Menfis as a parent are within thescope of this invention, including plants derived from hybrid cucumberMenfis.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which cucumber plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,leaves, stems, and the like.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed hybrid.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector includes DNA comprisinga gene under control of, or operatively linked to, a regulatory element(for example, a promoter). The expression vector may contain one or moresuch operably linked gene/regulatory element combinations. The vector(s)may be in the form of a plasmid, and can be used alone or in combinationwith other plasmids, to provide transformed cucumber plants, usingtransformation methods as described below to incorporate transgenes intothe genetic material of the cucumber plant(s).

Expression Vectors for Cucumber Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection (i.e., inhibiting growth of cells that do not containthe selectable marker gene), or by positive selection (i.e., screeningfor the product encoded by the genetic marker). Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signals,confers resistance to kanamycin (Fraley, et al., PNAS, 80:4803 (1983)).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin (Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant (Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.;7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil (Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); Stalker, et al., Science, 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); DeBlock, etal., EMBO J., 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available (Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991)). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie, et al., Science, 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Cucumber Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression incucumber. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in cucumber. With an inducible promoter therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See, Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991); Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)); or Tet repressor from Tn10 (Gatz,et al., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena,et al., PNAS, 88:0421 (1991)).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression incucumber or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in cucumber.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989);Christensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992); Atanassova, et al., Plant Journal, 2(3): 291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin cucumber. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in cucumber. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983); Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter, such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985); Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter, such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter, such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter,such as that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224(1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is cucumber. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

A. Genes that Confer Resistance to Pests or Disease and that Encode:

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Shoresh, et al., “Characterizationof a Mitogen-Activated Protein Kinase Gene from Cucumber Required forTrichoderma-Conferred Plant Resistance,” Plant Physiol., 142(3):1169-1179 (November 2006) (activation of Trichoderma-induced MAPK (TIPK)gene is necessary for the plant's Trichoderma-conferred defense againstbacterial pathogens); Jones, et al., Science, 266:789 (1994) (cloning ofthe tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin, etal., Science, 262:1432 (1993) (tomato Pto gene for resistance toPseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos, etal., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser, et al., Gene,48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes; and Atsushi, etal., “Possible Involvement of Leaf Gibberellins in the Clock-ControlledExpression of XSP30, a Gene Encoding a Xylem Sap Lectin, in CucumberRoots,” Plant Physiol., 133(4): 1779-1790 (December 2003).

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani, etal., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof; a mimetic based thereon, or anantagonist or agonist thereof See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor); and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins

8. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see, Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule. Forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Molec. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones; and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. See,Taylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J., 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

B. Genes that Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988); and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

2. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also Umaballava-Mobapathie in TransgenicResearch., 8: 1, 33-44 (1999) that discloses Lactuca sativa resistant toglufosinate. European Patent Application No. 0 333 033 to Kumada, etal., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides, such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentApplication No. 0 242 246 to Leemans, et al., DeGreef, et al.,Bio/technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc1-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

3. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

4. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

5. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication No. WO 01/12825.

C. Genes that Confer or Contribute to a Value-Added Trait, such as:

1. Increased iron content of the cucumber, for example, by transforminga plant with a soybean ferritin gene as described in Goto, et al., ActaHorticulturae, 521, 101-109 (2000).

2. Increased sweetness of the cucumber by transferring a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See, Penarrubia, et al., Bio/technology, 10: 561-564(1992). Specific regulatory elements involved in Glc repression alsohave been identified in the promotors of the cucumber malate synthase.See, Sarah, et al., (1996). “Distinct cis-acting elements direct thegermination and sugar responses of the cucumber malate synthase gene.”Mol. Gen. Genet., 250: 153-161.

3. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See, Knultzon, et al., PNAS, 89:2625 (1992).

4. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See, Shiroza, et al., J. Bacteriol.,170:810 (1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifonnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Sogaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See International Publication No. WO 01/29237.

2. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622, 1992).

E. Genes that Create a Site for Site Specific DNA Integration:

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep., (2003) 21:925-932 and WO99/25821, which are hereby incorporated by reference. Other systems thatmay be used include the Gin recombinase of phage Mu (Maeser, et al.,(1991); Vicki Chandler, The Maize Handbook, Ch. 118, Springer-Verlag(1994)), the Pin recombinase of E. coli (Enomoto, et al., (1983)), andthe R/RS system of the pSR1 plasmid (Araki, et al., (1992)).

F. Genes that Affect Abiotic Stress Resistance:

Genes that affect abiotic stress resistance (including, but not limitedto, flowering, pod and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see, WO 00/73475, where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009; 5,965,705; 5,929,305;5,891,859; 6,417,428; 6,664,446; 6,706,866; 6,717,034; 6,801,104;International Publication Nos. WO 2000/060089; WO 2001/026459; WO2001/035725; WO 2001/034726; WO 2001/035727; WO 2001/036444; WO2001/036597; WO 2001/036598; WO 2002/015675; WO 2002/017430; WO2002/077185; WO 2002/079403; WO 2003/013227; WO 2003/013228; WO2003/014327; WO 2004/031349; WO 2004/076638; WO 98/09521; and WO99/38977 describing genes, including CBF genes and transcription factorseffective in mitigating the negative effects of freezing, high salinity,and drought on plants, as well as conferring other positive effects onplant phenotype; U.S. Publication No. 2004/0148654 and InternationalPublication No. WO 01/36596 where abscisic acid is altered in plantsresulting in improved plant phenotype such as increased yield and/orincreased tolerance to abiotic stress; International Publication Nos. WO2000/006341; WO 04/090143; U.S. application Ser. No. 10/817,483 and U.S.Pat. No. 6,992,237, where cytokinin expression is modified resulting inplants with increased stress tolerance, such as drought tolerance,and/or increased yield. Also see International Publication Nos. WO02/02776; WO 2003/052063; JP2002281975; U.S. Patent No. 6,084,153;International Publication No. WO 01/64898; U.S. Pat. Nos. 6,177,275 and6,107,547 (enhancement of nitrogen utilization and altered nitrogenresponsiveness). For ethylene alteration, see, U.S. Publication Nos.2004/0128719; 2003/0166197; and International Publication No. WO2000/32761. For plant transcription factors or transcriptionalregulators of abiotic stress, see, e.g., U.S. Publication Nos.2004/0098764 or 2004/0078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants. See, e.g.,International Publication Nos. WO 97/49811 (LHY); WO 98/56918 (ESD4); WO97/10339; U.S. Pat. No. 6,573,430 (TFL); U.S. Pat. No. 6,713,663 (FT);International Publication Nos. WO 96/14414 (CON); WO 96/38560; WO01/21822 (VRN1); WO 00/44918 (VRN2); WO 99/49064 (GI); WO 00/46358(FRI); WO 97/29123; U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI); andInternational Publication Nos. WO 99/09174 (D8 and Rht), WO 2004/076638,and WO 2004/031349 (transcription factors).

Methods for Cucumber Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45: 279, 1441-1449 (1994); Tones, et al., Plantcell Tissue and Organic Culture, 34: 3, 279-285 (1993); Dinant, et al.,Molecular Breeding, 3: 1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation, collectively referred to asdirect gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 μm to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s, which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Pl. Cell. Rep., 12(3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20(2,October), 357-359 (1992); Aragao, F. J. L., et al., Pl. Cell. Rep.,12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93: 142-150(1996); Kim, J., Minamikawa, T., Plant Science, 117: 131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985); Christou, et al., PNAS, 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985); Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4): 507-514(1997/98); Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell, 4:1495-1505 (1992); Spencer, et al., Plant Mol. Biol.,24:51-61 (1994). See also Chupean, et al., Bio/technology, 7: 5, 503-508(1989).

Following transformation of cucumber target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues, and/or plants usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossedwith another (non-transformed or transformed) line in order to produce anew transgenic cucumber line. Alternatively, a genetic trait which hasbeen engineered into a particular cucumber cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

C. Genetic Marker Profile Through SSR and First Generation Progeny:

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety, ora related variety, or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs), which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Cregan et al., “An Integrated Genetic Linkage Map of theSoybean Genome,” Crop Science, 39:1464-1490 (1999), and Berry, et al.,“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties,”Genetics, 165:331-342 (2003), each of which are incorporated byreference herein in their entirety.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forhybrid cucumber Menfis.

Primers and PCR protocols for assaying these and other markers aredisclosed in the Soybase (sponsored by the USDA Agricultural ResearchService and Iowa State University). In addition to being used foridentification of hybrid cucumber Menfis, and plant parts and plantcells of variety hybrid cucumber Menfis, the genetic profile may be usedto identify a cucumber plant produced through the use of hybrid cucumberMenfis or to verify a pedigree for progeny plants produced through theuse of hybrid cucumber Menfis. The genetic marker profile is also usefulin breeding and developing backcross conversions.

The present invention comprises a cucumber plant characterized bymolecular and physiological data obtained from the representative sampleof said variety deposited with the American Type Culture Collection(ATCC). Further provided by the invention is a cucumber plant formed bythe combination of the disclosed cucumber plant or plant cell withanother cucumber plant or cell and comprising the homozygous alleles ofthe variety.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. See, for example, Gong, L., et al., “Microsatellites for thegenus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepoL.,” Theor. Appl. Genet., 117(1): 37-48 (2008 June). Another advantageof this type of marker is that, through use of flanking primers,detection of SSRs can be achieved, for example, by the polymerase chainreaction (PCR), thereby eliminating the need for labor-intensiveSouthern hybridization. The PCR detection is done by use of twooligonucleotide primers flanking the polymorphic segment of repetitiveDNA. Repeated cycles of heat denaturation of the DNA followed byannealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties, it is preferable if all SSRprofiles are performed in the same lab.

A number of promoters have utility for plant gene expression for anygene of interest including, but not limited to, selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements, and any other gene of agronomic interest.Examples of constitutive promoters useful for cucumber plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel, et al., (1985)), includingmonocots (see, e.g., Dekeyser, et al., (1990); Terada and Shimamoto,(1990)); a tandemly duplicated version of the CaMV 35S promoter, theenhanced 35S promoter (P-e35S) the nopaline synthase promoter (An, etal., (1988)); the octopine synthase promoter (Fromm, et al., (1989));and the figwort mosaic virus (P-FMV) promoter as described in U.S. Pat.No. 5,378,619 and an enhanced version of the FMV promoter (P-eFMV) wherethe promoter sequence of P-FMV is duplicated in tandem, the cauliflowermosaic virus 19S promoter, a sugarcane bacilliform virus promoter, acommelina yellow mottle virus promoter, and other plant DNA viruspromoters known to express in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can beused for expression of an operably linked gene in plant cells, includingpromoters regulated by: (1) heat (Callis, et al., (1988)), (2) light(e.g., pea rbcS-3A promoter, Kuhlemeier, et al., (1989); maize rbcSpromoter, Schaffner and Sheen, (1991); or chlorophyll a/b-bindingprotein promoter, Simpson, et al., (1985)), (3) hormones, such asabscisic acid (Marcotte, et al., (1989)), (4) wounding (e.g., wunl,Siebertz, et al., (1989)), or (5) chemicals, such as methyl jasmonate,salicylic acid, or Safener. It may also be advantageous to employorgan-specific promoters (e.g., Roshal, et al., (1987); Schernthaner, etal., (1988); Bustos, et al., (1989)).

Exemplary nucleic acids which may be introduced to the cucumber lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with, or are presentin, the same species but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a cucumber plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a cucumber plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance, such as genes for fungal disease control,herbicide tolerance, such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology, or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including, but not limited to, a Bacillus insect controlprotein gene as described in WO 99/31248, herein incorporated byreference in its entirety, and U.S. Pat. Nos. 5,500,365, 5,689,052, and5,880,275, herein incorporated by reference it their entirety. Inanother embodiment, the structural gene can confer tolerance to theherbicide glyphosate as conferred by genes including, but not limitedto, Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4)as described in U.S. Pat. No. 5,633,435, herein incorporated byreference in its entirety, or glyphosate oxidoreductase gene (GOX) asdescribed in U.S. Pat. No. 5,463,175, herein incorporated by referencein its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Bird,et al., (1991)). The RNA could also be a catalytic RNA molecule (i.e., aribozyme) engineered to cleave a desired endogenous mRNA product (see,for example, Gibson and Shillito, (1997)). Thus, any gene which producesa protein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of the present invention.

Single-Gene Conversions

When the terms cucumber plant, cultivar, hybrid, or cucumber line areused in the context of the present invention, this also includes anysingle gene conversions of that line. The term “single gene convertedplant” as used herein refers to those cucumber plants which aredeveloped by a plant breeding technique, called “backcrossing,” whereinessentially all of the desired morphological and physiologicalcharacteristics of a cultivar are recovered in addition to the singlegene transferred into the line via the backcrossing technique.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the line. The term “backcrossing” asused herein refers to the repeated crossing of a hybrid progeny back toone of the parental cucumber plants for that line, backcrossing 1, 2, 3,4, 5, 6, 7, 8, or more times to the recurrent parent. The parentalcucumber plant which contributes the gene for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental cucumber plant towhich the gene or genes from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol. Poehlman & Sleper, (1994); Fehr, (1987). In atypical backcross protocol, the original cultivar of interest (recurrentparent) is crossed to a second line (nonrecurrent parent) that carriesthe single gene of interest to be transferred. The resulting progenyfrom this cross are then crossed again to the recurrent parent and theprocess is repeated until a cucumber plant is obtained whereinessentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross. One ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic. Examples of these traits include, but are not limited to,male sterility, modified fatty acid metabolism, modified carbohydratemetabolism, herbicide resistance, resistance for bacterial, fungal, orviral disease, insect resistance, enhanced nutritional quality,industrial usage, yield stability, and yield enhancement. These genesare generally inherited through the nucleus. Several of these singlegene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957, and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of cucumber andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27: 9,1030-1032 (1992); Teng, et al., HortScience; 28: 6, 669-1671 (1993),Zhang, et al., Journal of Genetics and Breeding, 46: 3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38: 1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45: 279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125: 6, 669-672 (2000); and Ibrahim, et al.,Plant Cell, Tissue and Organ Culture, 28(2): 139-145 (1992). It is clearfrom the literature that the state of the art is such that these methodsof obtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce cucumber plants having thephysiological and morphological characteristics of hybrid cucumberMenfis.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, and the like. Means forpreparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Tissue culture of cucumber can be used for the in vitro regeneration ofcucumber plants. Tissues cultures of various tissues of cucumber andregeneration of plants therefrom are well-known and published. By way ofexample, tissue cultures, some comprising organs to be used to produceregenerated plants, have been described in Burza, et al., PlantBreeding, 114: 4, 341-345 (1995); Cui Hongwen, et al., Cucurbit GeneticsCooperative Report, 22, 5-7 (1999); Pellinen, Angewandte Botanik, 71:3/4, 116-118 (1997); Kuijpers, et al., Plant Cell Tissue and OrganCulture, 46: 1, 81-83 (1996); Colijn-Hooymans, et al., Plant Cell Tissueand Organ Culture, 39: 3, 211-217 (1994); Lou, et al., HortScience, 29:8, 906-909 (1994); Tabei, et al., Breeding Science, 44: 1, 47-51 (1994);Sarmanto, et al., Plant Cell Tissue and Organ Culture, 31:3 185-193(1992); Raharjo, et al., Cucurbit Genetics Cooperative Report, 15, 35-39(1992); Garcia-Sobo, et al., Cucurbit Genetics Cooperative Report, 15,40-44 (1992); Cade, et al., Journal of the American Society forHorticultural Science, 115:4 691-696 (1990); Chee, et al., HortScience,25:7, 792-793 (1990); Kim, et al., HortScience, 24:4 702 (1989); Punja,et al., Plant Cell Rep., 9:2 61-64 (1990). It should also be mentionedthat the regeneration of the cucumber after induction of adventitiousshoot buds on calli derived from cotyledons, has been described inMsikita, et al., Cucurbit Genetics Cooperative Report, 11: 5-7 (1988),Kim, et al., Plant Cell Tissue Organ Culture, 12: 67-74 (1988); Wehner,et al., HortScience, 16: 759-760 (1981) had previously described theinduction of buds on cotyledons. Cucumber plants could be regenerated bysomatic embryogenesis. These somatic embryos developed either in cellsuspensions derived from calli developed from leaf explants (Chee, etal., Plant Cell Report, 7: 274-277 (1988)) or hypocotyls (Rajasekaran,et al., Annals of Botany, 52: p. 417-420 (1983)), or directly oncotyledonous (Cade, et al., Cucurbit Genetics Cooperative Report, 11:3-4(1988)) or leaf calli (Malepszy, et al., Pfanzenphysiologie, 111:273-276 (1983)). It is clear from the literature that the state of theart is such that these methods of obtaining plants are “conventional” inthe sense that they are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce cucumber plants having thephysiological and morphological characteristics of hybrid cucumberMenfis.

Additional Breeding Methods

This invention also is directed to methods for producing a cucumberplant by crossing a first parent cucumber plant with a second parentcucumber plant wherein the first or second parent cucumber plant is acucumber plant of Menfis. Further, both first and second parent cucumberplants can come from hybrid cucumber Menfis. Thus, any such methodsusing hybrid cucumber Menfis are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using hybrid cucumber Menfis as at least one parentare within the scope of this invention, including those developed fromcultivars derived from hybrid cucumber Menfis. Advantageously, thiscucumber plant could be used in crosses with other, different, cucumberplants to produce the first generation (F₁) cucumber hybrid seeds andplants with superior characteristics. The cultivar of the invention canalso be used for transformation where exogenous genes are introduced andexpressed by the cultivar of the invention. Genetic variants createdeither through traditional breeding methods using hybrid cucumber Menfisor through transformation of hybrid cucumber Menfis by any of a numberof protocols known to those of skill in the art are intended to bewithin the scope of this invention.

The following describes breeding methods that may be used with hybridcucumber Menfis in the development of further cucumber plants. One suchembodiment is a method for developing hybrid cucumber Menfis progenycucumber plants in a cucumber plant breeding program comprising:obtaining the cucumber plant, or a part thereof, of hybrid cucumberMenfis, utilizing said plant or plant part as a source of breedingmaterial, and selecting a cucumber Menfis progeny plant with molecularmarkers in common with hybrid cucumber Menfis and/or with morphologicaland/or physiological characteristics selected from the characteristicslisted in Table 1. Breeding steps that may be used in the cucumber plantbreeding program include pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers) and the making of double haploidsmay be utilized.

Another method involves producing a population of hybrid cucumber Menfisprogeny cucumber plants, comprising crossing hybrid cucumber Menfis withanother cucumber plant, thereby producing a population of cucumberplants, which, on average, derive 50% of their alleles from hybridcucumber Menfis. A plant of this population may be selected andrepeatedly selfed or sibbed with a cucumber plant resulting from thesesuccessive filial generations. One embodiment of this invention is thecucumber plant produced by this method and that has obtained at least50% of its alleles from hybrid cucumber Menfis.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus, the invention includes hybridcucumber Menfis progeny cucumber plants comprising a combination of atleast two Menfis traits selected from the group consisting of thoselisted in Table 1 or the Menfis combination of traits listed in theSummary of the Invention, so that said progeny cucumber plant is notsignificantly different for said traits than cucumber Menfis asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or may be characterized by percentsimilarity or identity to hybrid cucumber Menfis as determined by SSRmarkers. Using techniques described herein, molecular markers may beused to identify said progeny plant as a hybrid cucumber Menfis progenyplant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such avariety is developed its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of hybrid cucumber Menfis may also be characterized throughtheir filial relationship with hybrid cucumber Menfis, as, for example,being within a certain number of breeding crosses of hybrid cucumberMenfis. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween hybrid cucumber Menfis and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4, or 5breeding crosses of hybrid cucumber Menfis.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which cucumber plants canbe regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,seeds, stems, and the like.

Table 2 compares characteristics for Menfis with Onofre.

TABLE 2 Characteristic Menfis Onofre Leaf Dark green leaves Light greenleaves Cucumber yellow stunting Intermediate resistant Susceptibledisorder virus

Deposit Information

A deposit of the hybrid cucumber of this invention is maintained by EnzaZaden USA, Inc., having an address at 7 Harris Place, Salinas, Calif.93901. Access to this deposit will be available during the pendency ofthis application to persons determined by the Commissioner of Patent andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110 or National Collections ofIndustrial, Food and Marine Bacteria (NCIMB), 23 St. Machar Drive,Aberdeen, Scotland, AB24 3RY, United Kingdom.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A hybrid cucumber seed designated Menfis whereina representative sample of seed has been deposited under ATCC AccessionNo. ______.
 2. A cucumber plant, or a part thereof, produced by growingthe seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule ofthe plant of claim
 2. 5. A protoplast produced from the plant of claim2.
 6. A tissue culture produced from protoplasts or cells from the plantof claim 2, wherein said cells or protoplasts are produced from a plantpart selected from the group consisting of leaf, anther, pistil, stem,petiole, root, root tip, fruit, seed, flower, cotyledon, hypocotyl,embryo, and meristematic cell.
 7. A cucumber plant regenerated from thetissue culture of claim
 6. 8. A protoplast produced from the tissueculture of claim
 6. 9. A method for producing a hybrid cucumber seedcomprising crossing the cucumber plant of claim 2 with a differentcucumber plant and harvesting the resultant hybrid cucumber seed.
 10. Ahybrid cucumber seed produced by the method of claim
 9. 11. A hybridcucumber plant, or a part thereof, produced by growing said hybridcucumber seed of claim
 10. 12. The method of claim 9, wherein at leastone of said cucumber plants is transgenic.
 13. A method of producing anherbicide resistant cucumber plant, wherein said method comprisedintroducing a gene conferring herbicide resistance into the plant ofclaim
 2. 14. An herbicide resistant cucumber plant produced by themethod of claim 13, wherein the gene confers resistance to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, dicamba, phenoxy proprionic acid, glufosinate,L-phosphinothricin, triazine, and benzonitrile.
 15. A method ofproducing a pest or insect resistant cucumber plant, wherein said methodcomprises introducing a gene conferring pest or insect resistance intothe cucumber plant of claim
 2. 16. A pest or insect resistant plantproduced by the method of claim
 15. 17. The cucumber plant of claim 16,wherein the transgene encodes a Bacillus thuringiensis (Bt) endotoxin.18. A method of producing a cucumber plant wherein the method comprisesintroducing the cucumber plant of claim 2 with a transgene.
 19. Acucumber plant produced by the method of claim
 18. 20. A method ofproducing a cucumber plant with modified fatty acid metabolism ormodified carbohydrate metabolism wherein the method comprisestransforming the cucumber plant of claim 2 with a transgene encoding aprotein selected from the group consisting of fructosyltransferase,levansucrase, alpha-amylase, invertase, and starch branching enzyme orencoding an antisense of stearyl-ACP desaturase.
 21. A cucumber planthaving modified fatty acid metabolism or modified carbohydratemetabolism produced by the method of claim 20.